Pettus Benjamin J, Bielawski Jacek, Porcelli Anna M, Reames Davis L, Johnson Korey R, Morrow Jason, Chalfant Charles E, Obeid Lina M, Hannun Yusuf A
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
FASEB J. 2003 Aug;17(11):1411-21. doi: 10.1096/fj.02-1038com.
In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor-alpha (TNF) induced prostaglandin E2 (PGE2) production by 4 h and cyclooxygenase-2 (COX-2) induction as early as 2 h. This TNF-induced PGE2 production was inhibited by NS398, a COX-2 selective inhibitor. GC-MS analysis revealed that only COX-2-generated prostanoids were produced in response to TNF, thus providing further evidence of COX-2 selectivity. As sphingolipids have been implicated in mediating several actions of TNF, their role in COX-2 induction and PGE2 production was evaluated. Sphingosine-1-phosphate (S1P) induced both COX-2 and PGE2 in a dose-responsive manner with an apparent ED50 of 100-300 nM. The related sphingolipid sphingosine also induced PGE2, though with much less efficacy. TNF induced a 3.5-fold increase in sphingosine-1-phosphate levels at 10 min that rapidly returned to baseline by 40 min. Small interfering RNAs (siRNAs) directed against mouse SK1 decreased (typically by 80%) SK1 protein and inhibited TNF-induced SK activity. Treatment of cells with RNAi to SK1 but not SK2 almost completely abolished the ability of TNF to induce COX-2 or generate PGE2. By contrast, cells treated with RNAi to S1P lyase or S1P phosphatase enhanced COX-2 induction leading to enhanced generation of PGE2. Treatment with SK1 RNAi also abolished the effects of exogenous sphingosine and ceramide on PGE2, revealing that the action of sphingosine and ceramide are due to intracellular metabolism into S1P. Collectively, these results provide novel evidence that SK1 and S1P are necessary for TNF to induce COX-2 and PGE2 production. Based on these findings, this study indicates that SK1 and S1P could be implicated in pathological inflammatory disorders and cancer.
在本研究中，我们探讨了鞘脂代谢在炎症反应中的作用。在L929成纤维细胞模型中，肿瘤坏死因子-α（TNF）在4小时内诱导前列腺素E2（PGE2）生成，最早在2小时诱导环氧合酶-2（COX-2）。这种TNF诱导的PGE2生成被COX-2选择性抑制剂NS398抑制。气相色谱-质谱分析显示，仅COX-2产生的前列腺素类物质在响应TNF时产生，从而提供了COX-2选择性的进一步证据。由于鞘脂已被认为介导TNF的多种作用，因此评估了它们在COX-2诱导和PGE2生成中的作用。鞘氨醇-1-磷酸（S1P）以剂量反应方式诱导COX-2和PGE2，表观半数有效剂量（ED50）为100 - 300 nM。相关的鞘脂鞘氨醇也诱导PGE2，尽管效力要低得多。TNF在10分钟时使鞘氨醇-1-磷酸水平增加3.5倍，到40分钟时迅速恢复到基线。针对小鼠SK1的小干扰RNA（siRNA）降低（通常降低80%）SK1蛋白并抑制TNF诱导的SK活性。用RNAi处理细胞以靶向SK1而非SK2几乎完全消除了TNF诱导COX-2或生成PGE2的能力。相比之下，用RNAi处理细胞以靶向S1P裂解酶或S1P磷酸酶增强了COX-2诱导，导致PGE2生成增加。用SK1 RNAi处理也消除了外源性鞘氨醇和神经酰胺对PGE2产生的影响，表明鞘氨醇和神经酰胺的作用是由于细胞内代谢为S1P。总体而言，这些结果提供了新的证据，表明SK1和S1P是TNF诱导COX-2和PGE2生成所必需的。基于这些发现，本研究表明SK1和S1P可能与病理性炎症疾病和癌症有关。
